Human multipotent stromal cells attenuate lipopolysaccharide-induced acute lung injury in mice via secretion of tumor necrosis factor-α-induced protein 6
© Danchuk et al.; licensee BioMed Central Ltd. 2011
Received: 21 December 2010
Accepted: 13 May 2011
Published: 13 May 2011
Multipotent stromal cells (MSCs) are currently in clinical trials for a number of inflammatory diseases. Recent studies have demonstrated the ability of MSCs to attenuate inflammation in rodent models of acute lung injury (ALI) suggesting that MSCs may also be beneficial in treating ALI.
To better understand how human MSCs (hMSCs) may act in ALI, the lungs of immunocompetent mice were exposed to lipopolysaccharide (LPS) and four hours later bone marrow derived hMSCs were delivered by oropharyngeal aspiration (OA). The effect of hMSCs on lung injury was assessed by measuring the lung wet/dry weight ratio and total protein in bronchoalveolar lavage (BAL) fluid 24 or 48 h after LPS. BAL fluid was also analyzed for the presence of inflammatory cells and cytokine expression by multiplex immunoassay. Microarray analysis of total RNA isolated from treated and untreated lungs was performed to elucidate the mechanism(s) involved in hMSC modulation of lung inflammation.
Administration of hMSCs significantly reduced the expression of pro-inflammatory cytokines, neutrophil counts and total protein in bronchoalveolar lavage. There was a concomitant reduction in pulmonary edema. The anti-inflammatory effects of hMSCs were not dependent on localization to the lung, as intraperitoneal administration of hMSCs also attenuated LPS-induced inflammation in the lung. Microarray analysis revealed significant induction of tumor necrosis factor (TNF)-α-induced protein 6 (TNFAIP6/ TSG-6) expression by hMSCs 12 h after OA delivery to LPS-exposed lungs. Knockdown of TSG-6 expression in hMSCs by RNA interference abrogated most of their anti-inflammatory effects. In addition, intra-pulmonary delivery of recombinant human TSG-6 reduced LPS-induced inflammation in the lung.
These results show that hMSCs recapitulate the observed beneficial effects of rodent MSCs in animal models of ALI and suggest that the anti-inflammatory properties of hMSCs in the lung are explained, at least in part, by activation of hMSCs to secrete TSG-6.
Acute lung injury (ALI) and its more severe manifestation, acute respiratory distress syndrome (ARDS), are major complications in critically ill patients. ALI is a syndrome of widespread lung inflammation and increased pulmonary vascular permeability resulting in pulmonary edema, hypoxia and may contribute to multiple organ failure and death. ALI/ARDS is most commonly caused by sepsis, pneumonia, trauma or aspiration of gastric contents. Despite improvements in critical care and mechanical ventilation protocols, the mortality rate for patients with ALI is still 30% to 40% [1–3]. Multipotent stromal cells (MSCs), also known as mesenchymal stem cells, have been proposed as a cellular therapy for ALI.
MSCs are fibroblast-like cells characterized by their ability to self-renew and undergo differentiation into mesenchymal lineage cell types including bone, cartilage, adipose tissue, muscle and tendon . MSCs have been isolated from the bone marrow and the connective tissue of almost all organs including adipose, periosteum, synovial fluid, muscle, hair follicles, root of deciduous teeth, articular cartilage, placenta, dermis, umbilical cord, Wharton's jelly, lung, liver and spleen [4–6]. The interest in MSCs as cellular therapy arises from numerous in vivo studies showing that MSCs avoid allorecognition, home to sites of injury, and suppress inflammation as well as immune responses. In addition, MSCs can be rapidly expanded in vitro while maintaining their multipotent properties . MSCs are currently in clinical trials for treatment of a number of human diseases including osteogenesis imperfecta, osteoarthritis, graft-versus-host disease, multiple sclerosis, types 1 and 2 diabetes, Crohn's disease, acute kidney injury, acute myocardial infarction, ischemic heart failure, and chronic obstructive pulmonary disease .
Intravenous or intra-alveolar administration of MSCs modulates both the inflammatory process and tissue remodeling in experimental models of ALI despite minimal, if any, engraftment (reviewed in ). The mechanisms involved are poorly understood; however, increasing data suggest that the protective effects of MSCs are largely mediated through production of paracrine mediators [7, 10]. Most of what is known about the therapeutic function of MSCs comes from studies that employed rodent MSCs. It has been suggested that the mechanisms of MSC-meditated immunosuppression may be different between rodent MSCs and human MSCs (hMSCs) in some models  but not in others, including the experimental allergic encephalomyelitis model for multiple sclerosis [12, 13]. Before proceeding to clinical trials, it is critical to understand the mechanisms integral to the beneficial effects of hMSCs in ALI so that the human physiological response can be accurately predicted.
To elucidate the mechanisms involved in human MSC modulation of inflammation in the lung, we used intra-pulmonary delivery of E. coli endotoxin to immunocompetent mice, a well-characterized model of ALI. Here we demonstrated that xenographic transplantation of hMSCs suppress inflammation and lung injury. Taking advantage of the cross-species nature of our experiments, we employed human-specific gene arrays to identify potential anti-inflammatory factors expressed by hMSCs in the injured lung. TNF-α-induced protein 6 (TNAIP6/ TSG-6) was shown to be highly induced in hMSCS in response to lung injury. Using RNA interference, we demonstrated that the anti-inflammatory properties of hMSCs in the lung are explained, at least in part, by activation of hMSCs to secrete TSG-6.
Materials and methods
Adult hMSCs from bone marrow were obtained from the Center for the Preparation and Distribution of Adult Stem Cells . The center has supplied standardized preparations of MSCs enriched for early progenitor cells to over 350 laboratories under the auspices of a National Institutes of Health/National Center for Research Resources (NIH/NCRR) grant (P40 RR17447). Vials of about 0.5 × 106 cells (passage 1 or 2) were thawed, plated on two-stack culture chambers (Corning, Lowell, MA, USA) in 100 ml/stack of complete culture medium (CCM) at 150 cells/cm2 density, and incubated at 37°C in 5% CO2 for six to seven days until 70% confluent. The CCM consisted of α-MEM (Invitrogen, Gaithersburg, MD, USA) containing 17% FBS (lot-selected for rapid growth of hMSCs; Atlanta Biologicals, Lawrenceville, GA, USA); 100 units/ml of penicillin; 100 μg/ml of streptomycin; and 2 mM L-glutamine (Invitrogen). One day before cells were to be administered, medium was changed to antibiotic-free CCM. hMSCs were harvested with 0.25% trypsin/1 mM EDTA (Invitrogen) for 3 minutes at 37°C, washed with PBS (without Ca2+ and Mg2+) by centrifugation at 480 × g for 10 minutes at room temperature, resuspended in PBS and administered to mice. IMR-90 human lung fibroblasts (HLFs) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured according to manufacturer's instructions. HLFs were used at passages 14 to 15 for all experiments.
Animal model of ALI
Animal care and all animal procedures were approved by the Institutional Animal Care and Use Committee of Tulane University. Eight- to-ten-week-old female BALB/C mice (National Cancer Institute-Frederick, Frederick, MD, USA) were treated with either 1 mg/kg lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA) from Escherichia coli (serotype 0111:B4) in 100 μl PBS or an equal volume of PBS, as vehicle control, by oropharyngeal aspiration (OA) as previously described . Briefly, mice were anesthetized with 2% isoflurane vapor (VetOne, Meridian, ID, USA) in oxygen and suspended on a string by the cranial incisors. A droplet was placed in the back of the throat while simultaneously holding the tongue to block the swallow reflex and pinching the nose, forcing the mouse to breathe through its mouth and in the process aspirate the liquid. Four hours after LPS exposure, hMSCs or HLFs (2.5 × 105/100 μl PBS) were given by OA and 30 minutes later a second dose of equal concentration was administered, for a total of 5 × 105 cells. As the control, 200 μl PBS was delivered as described above. Recombinant human TSG-6 protein (rhTSG-6, R&D Systems, Minneapolis, MN, USA) was administered at 50 μg/200 μl PBS by OA in two equal doses, 30 minutes apart, 1 h and 10 h after LPS delivery to the lung. Mice were anesthetized with 80 mg/kg ketamine plus 8 mg/kg xylazine and euthanized by exsanguination 24 or 48 h after LPS treatment. The right lung was processed for bronchoalveolar lavage (BAL) and the left lung was used for RNA isolation or histology. Dry/wet weight measurements were performed on whole lungs from a separate set of animals.
Lungs were perfused with 10% buffered formalin (Sigma-Aldrich) at a pressure of 25 cm H2O for 15 to 20 minutes, removed from the animal and placed in fresh 10% neutral buffered formalin for 16 to 20 h at 4°C prior to processing and embedding. Sections (4 μM) from each sample were stained with hematoxylin and eosin (H&E) for histopathological evaluation by two independent experts blinded to the treatment. Whole H&E-stained sections were digitally imaged using an Aperio ScanScope (Aperio Technologies, Vista, CA, USA). TIFF Images of whole lung sections were acquired using the ImageScope program from Aperio. Threshold analysis of whole lung sections, excluding non-parenchymal structures (for example, large blood vessels, airway structural elements, connective structures, and so on), was performed using the NIH ImageJ program. Briefly, RGB color images were split into individual color channels (RGB stack), and analyses were performed using the green channel. The threshold was then adjusted to the highest point that would highlight all areas of the parenchyma while excluding background. The threshold area of total parenchyma was measured and recorded. Then, the threshold was reset to highlight only areas of high pixel density which correlated with lung damage (damage threshold) as determined by microscopic regional image analysis (Supplemental Figure S1 in Additional file 1). Equal threshold values were applied across groups to all images analyzed. Lung injury index was calculated as the ratio of damaged area relative to total parenchyma area and reported as fold increase over control lung (PBS + PBS).
Bronchoalveolar lavage (BAL) and differential cell counts
Lung lavage was performed immediately after euthanasia with five aliquots consisting of 600 μl of sterile PBS supplemented with 0.4 mM EDTA and protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Aliquots were centrifuged at 1,500 × g for five minutes at 4°C to separate cells from supernatant. Supernatant from the first aliquot was stored at -70°C for biochemical analysis and cells from all aliquots were combined and counted using a grid hemocytometer. Differential cell counts were performed after cytocentrifugation and staining with a modified Wright stain (Diff-Quik, Fisher Scientific, Pittsburgh, PA, USA) by counting 300 cells in each of three different viewing fields.
Lung wet/dry weight ratio
Lungs were dissected immediately after euthanasia and the wet weight was recorded. Lungs were then placed in an incubator at 65°C for 72 h, and the dry weight was determined.
Cytokine and protein measurements
Measurement of cytokines and chemokines in BAL fluid was performed by multiplex immunoassay using Milliplex mouse cytokine/chemokine 22-plex kit (Millipore, Billerica, MA, USA) following the manufacturer's instructions. MIP-2 and MCP-1 levels were measured with mouse specific Quantikine CXCL2/MIP-2 and CCL2/JE/MCP-1 immunoassays, respectively (R&D Systems). The quantification of mouse IL-6 was performed with mouse IL-6 ELISA MAX™ Deluxe Set from BioLegend (San Diego, CA, USA) according to the manufacturer's instruction. Protein concentration was quantified using the Micro BCA protein assay kit (Pierce, Rockford, IL, USA).
RNA isolation and RT-PCR
Total RNA was obtained from lung tissue homogenized in TriPure Isolation Reagent (Roche) and was purified with the RNeasy mini kit (Qiagen, Valencia, CA, USA). Total RNA concentration was measured using NanoDrop spectrophotometer (Thermo Scientific Nanodrop, Nanodrop Technologies, Wilmington, DE, USA) and quality assessed by the 260/280 and 230/260 ratios. Reverse transcription was carried out with 1 μg of total RNA using a First Strand cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA) following the instructions provided by the manufacturer. Quantitative real-time PCR (qRT-PCR) was performed using a 40-cycle two-step PCR (95°C for 15 sec followed by 60°C for 1 minute) with sequence-specific primer pairs using the iCycler IQ real-time detection System (Bio-Rad). qRT-PCR for human TSG-6 and GAPDH mRNA was performed using human-specific TSG-6 primers/probe  or human GAPDH Taq-Man Gene Expression Assay (Hs00266705_g1, Applied Biosystems, Foster City, CA, USA) and Taqman Universal PCR Master Mix (Applied Biosystems) with 200 ng of cDNA. The number of viable hMSCs in mouse lung at the time of euthanasia was evaluated as a percentage of administered hMSCs based on qRT-PCR results for human GAPDH mRNA as previously described . Human keratinocyte growth factor (KGF) and interleukin 1 receptor antagonist (IL1RN) mRNA levels were assayed using Taq-Man Gene Expression Assays Hs00384281_m1 and Hs00893625_m1, respectively (Applied Biosystems). The species specificity of each primer set was confirmed by analyzing RNA isolated from mouse lung and hMSCs in each reaction.
RNA was isolated from whole lungs of LPS- (LPS + hMSC) or PBS-exposed (PBS + hMSC) mice 12 h after OA administration of 5 × 105 hMSCs. Control RNA was isolated from LPS-exposed lung treated with PBS (LPS + PBS). To establish baseline levels of gene expression in hMSCs prior to delivery to the lung, 5 × 105 hMSCs were added to LPS- (LPS + hMSCs in vitro mix) or PBS- (PBS + hMSCs in vitro mix) exposed mouse lung immediately prior to homogenization. RNA extracts from samples containing hMSCs were analyzed for human GAPDH mRNA by qRT-PCR to estimate the amount of human RNA in each sample. To allow comparisons between samples containing both human and mouse RNA, RNA extracts were diluted to contain equal levels of human RNA. The adjusted RNA samples (approximately 3 μg of total RNA) were assayed on a human (HG-U133 Plus 2.0) microarray (Affymetrix, Santa Clara, CA, USA). An aliquot of total RNA extracted from LPS-exposed lung treated with PBS, equal in concentration to the adjusted RNA sample from LPS-exposed lung treated with hMSCs, was also included to detect cross-hybridization of mouse RNA to the human chip. The data were analyzed using Partek Genomics Suite 6.5 (Partek Inc., St. Louis, MO, USA) and filtered for cross-hybridization (CV >0.5 and call >33%) as previously described . Values were expressed as fold changes relative to the signal intensities of controls. For a human gene to be considered significantly up-regulated in the LPS + hMSC sample the expression in the human array had to be at least two-fold higher than in the 1) LPS + PBS sample, 2) PBS + hMSC sample, 3) LPS + hMSC in vitro mix sample, and 4) PBS + hMSC in vitro mix sample.
Target hMSCs for transfection were cultured in antibiotic-free CCM and harvested as described above. About 1 × 106 hMSCs were resuspended in 100 μl resuspension buffer R (Neon Transfection System, Invitrogen, Carlsbad, CA, USA) with 1 μM siRNA for TSG-6 (sc-39819; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or control non-silencing siRNA (sc-37007; Santa Cruz Biotechnology) and transfected in 100 μl Neon tip with Neon transfection system (Invitrogen) using two pulses (1,400 V input pulse voltage/20 ms input pulse width). Transfected hMSCs were plated on 10 cm2 tissue culture dishes in 7.5 ml antibiotic-free CCM for 24 h.
Myeloperoxidase (MPO) activity
MPO activity in BAL fluid was measured by continually monitoring the change in absorbance of MPO reactions at 460 nm for a period of five minutes using the Synergy HT multi-detection microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Assays were conducted at 25°C in a final volume of 100 μl. The reaction buffer contained 50 mM potassium phosphate (pH 6.0), 0.0005% (volume/volume) H2O2 and 0.167 mg/ml o-dianisidine dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA). MPO activity in BAL fluid was expressed in units/min/ml using a standard curve with 0 to 0.625 units/ml human leukocyte myeloperoxidase (Sigma-Aldrich).
Data are shown as mean ± SEM. Comparisons between the two groups were made using unpaired, two-tailed Student's t tests. P < 0.05 was considered statistically significant. To assess the likely contribution of Type 1 error due to multiple analyses performed in the multiplex immunoassays, we calculated P- values for the null hypothesis based on the exact confidence intervals for samples from the binomial distribution [17, 18].
To elucidate the role of hMSCs in ALI, the lungs of immunocompetent mice were exposed to LPS and 4 h later 5 × 105 hMSCs, or HLFs as control, were delivered by OA. At 24 and 48 h after LPS, animals were euthanized by exanguination and lungs processed either for BAL, RNA extraction, histology or lung wet/dry weight measurements to assess inflammation and lung injury. All experiments were repeated at least two times with six animals per group, except where indicated.
Effect of hMSCs on LPS-induced pulmonary edema and microvascular permeability
Pulmonary edema and total protein in BAL fluid were assessed 24 h and 48 h after LPS as measures of lung injury and vascular leak. LPS-exposed mice displayed substantial pulmonary edema as indicated by an increased lung wet/dry weight ratio (P < 0.0007). Treatment with hMSCs significantly decreased the pulmonary edema at both 24 and 48 h (P < 0.05) (Figure 1c). A similar pattern was seen in the total protein concentrations in BAL fluid (Figure 1d) consistent with a decrease in microvascular permeability. Administration of control HLFs had no effect on lung wet/dry weight ratio or BAL fluid protein concentration (Figure 1c, d).
Effects of hMSCs on inflammation
hMSC upregulate expression of anti-inflammatory genes in response to inflammation
The top 10 transcripts of hMSCs, up-regulated in lung after OA delivery to LPS exposed mice
LPS + hMSCs vs LPS + hMSCs in vitro mix
LPS + hMSCs vs PBS + hMSCs
LPS + hMSCs vs LPS + PBS
Interferon-induced protein with tetratricopeptide repeats 3
Tumor necrosis factor, alpha-induced protein 6
Chemokine (C-X-C motif) ligand 6
Superoxide dismutase 2, mitochondrial
Chemokine (C-X-C motif) ligand 5
Spermidine/spermine N1-acetyltransferase 1
Pregnancy-associated plasma protein A, pappalysin 1
ATP-binding cassette, sub-family A, member 1
MSC expression of keratinocyte growth factor (KGF) was previously shown to be essential for the therapeutic effects of hMSCs in an ex vivo perfused lung model of ALI . No change in expression of KGF was detected by the microarray analysis performed here. However, qRT-PCR analysis of the RNA samples used for the microarrays demonstrated an approximately 11-fold induction in KGF expression in LPS + hMSCs exposed lung. hMSCs delivered to a PBS-exposed lung did not upregulate expression of KGF (Figure 5b).
Interleukin 1 receptor antagonist (IL-1RN), has been implicated in the anti-inflammatory function of MSCs . IL-1RN mRNA levels were not detectable by qRT-PCR in hMSCs prior to delivery to the lung (PBS + hMSCs in vitro mix). However, hMSCs in the PBS-exposed lungs expressed low levels of IL-1RN mRNA (CT's of 35 to 37) and hMSCs in LPS-exposed lungs expressed approximately five-fold higher levels of IL-1RN mRNA (Figure 5c).
Effect of TSG-6 on lung inflammation
Human MSCs attenuated LPS-induced lung inflammation and injury in immunocompetent mice. In response to the pro-inflammatory environment in the LPS exposed lung, hMSCs upregulated expression of a number of genes. Some of these genes encode secreted factors with known anti-inflammatory properties. One of the most highly upregulated genes was TNFAIP6 that encodes TSG-6. Knockdown of TSG-6 mRNA expression in hMSCs abrogated most of their anti-inflammatory effects. In addition, intra-pulmonary delivery of rhTSG-6 reduced LPS-induced inflammation in the lung. These results suggest that hMSCs in the lung upregulated expression of TNFAIP6 and the secreted TSG-6 decreased neutrophil accumulation and lung injury.
Previous studies have demonstrated the ability of murine MSCs to attenuate inflammation and lung injury in rodent models of ALI [19, 24, 25]. Recently, the ability of human MSCs to restore alveolar epithelial fluid transport and lung fluid balance was demonstrated in a study employing an ex vivo perfused human lung preparation injured by E. coli endotoxin . These studies have ignited much interest in hMSCs as cellular therapy for ALI. However, little is known about the mechanisms by which MSCs modulate lung inflammation and repair. The anti-inflammatory effects of mouse MSCs in the lung have been associated with MSC secretion of interleukin 1 receptor antagonist (IL1RN)  and TGF-β1. It is not clear how closely mMSCs resemble hMSCs. Certainly these two populations of cells share many features; however, there is some evidence that mMSCs and hMSCs differ in their response to inflammatory cytokines. For example, Ren et al. showed in vitro that inflammatory cytokines induce the expression of indoleamine 2,3-dioxygenase (IDO), but not inducible nitric oxide synthase (iNOS), in human MSCs, whereas in mouse MSCs the opposite is true . The goal of our study was to investigate the mechanism(s) by which human MSCs modulate inflammation in the lung.
For these studies we delivered human MSCs to immunocompetent mice after intrapulmonary delivery of LPS, a well-characterized mouse model of direct lung injury that recapitulates many of the features of human ALI including patchy intra-alveolar neutrophil infiltrates and changes in epithelial permeability. A similar cross-species strategy was used to elucidate the mechanism(s) by which hMSCs improve heart function after myocardial infarction . In that study, hMSCs were delivered to immunodeficient mice where they provided numerous endogenous markers for the cells and caused no obvious cross-species artifacts.
Our results demonstrated that hMSCs improved lung histology and reduced pulmonary edema in immunocompetent mice (Figure 1) similar to what has been reported for syngeneic mMSCs in this model [19, 24]. The improvement in lung injury was associated with a decrease in proinflammatory cytokines and chemokines, including some (IL-1α, IL-1β, MIP-1α and MIP-2) that function as chemoattractants and activators of neutrophils (Figures 2d and 3). Consistent with these results, hMSC treated lungs had significantly fewer neutrophils, the major mediator of lung injury in this model (Figure 2b). TNF-α levels in BAL fluid were not changed with hMSC treatment and although there was a trend toward increased IL-10 in BAL fluid the levels of IL-10 were very low (Supplemental Table S1 in Additional file 2). Administration of HLFs had no beneficial effects (Figure 2 and Supplemental Figure S2 in Additional file 3). These results suggest that hMSCs protected the lung primarily by preventing recruitment and/or activation of neutrophils. In a study similar to ours, mouse MSCs given four hours after LPS attenuated lung injury and reduced the levels of TNF-α and MIP-2 in BAL fluid. However, neutrophil influx or activation was not ameliorated . The differences in the MSC effects on neutrophil influx may be explained by intrinsic differences in MSCs from humans versus mice, but could also be explained by differences in experimental conditions between the studies, including strain of mice used, dose of LPS and method of intra-pulmonary delivery.
The ability of human MSCs to attenuate lung inflammation and injury in an immunocompetent mouse suggests that the anti-inflammatory action of hMSCs in the lung is non-major histocompatibility complex (MHC)-restricted and further, that hMSCs are to some degree immune privileged due to their immune modulatory effects, as others have suggested . These results support the use of allogeneic hMSCs in patients.
Our finding that hMSCs attenuated lung inflammation when administered intravenously (Figure 4) was not surprising since most of the injected cells are initially trapped in the lung . However, this is the first report that MSCs delivered intraperitoneally attenuate inflammation in the lung (Figure 4b, c). The hMSCs did not travel to the lung after IP injection as indicated by the absence of human GAPDH mRNA in the lung one hour (Figure 4a) or six hours (data not shown) after injection. The fate of the cells is currently under investigation. The IP route of delivery was not as effective as OA and IV routes in reducing LPS-induced lung injury. For these experiments, we used a dose of hMSCs (5 × 105) that we determined to be optimal for OA delivery. It is possible that increasing the number of hMSCs administered IP would provide better protection. Alternatively, our results may suggest that the factor or factors responsible for hMSC suppression of inflammation can function systemically, whereas factors that enhance lung repair may work best when delivered locally. Regardless, these results strongly suggest that the beneficial properties of hMSCs are predominantly mediated through secretion of factors that act in paracrine fashion.
The upregulation of TNFAIP6 by hMSCs in response to the inflammatory milieu of the LPS-exposed lung (Table 1) was of special interest because TSG-6 has previously been shown to play a role in the beneficial effects of hMSCs . TSG-6 is a 35 kDa, secreted protein produced by many cell types in response to TNF-α and IL-1β [20, 21]. hMSCs in the LPS-exposed lung upregulated their TSG-6 mRNA expression more than 900-fold (Figure 5a). TSG-6 is composed mainly of contiguous Link and CUB modules that interact with a broad spectrum of glycosaminoglycans and proteins, including HA, chondroitin 4-sulphate, dermatan sulfate, heparan sulfate, heparin, inter-α-inhibitor, versican, aggrecan, thrombospondin-1, and PTX3 . The anti-inflammatory properties of TSG-6 are well documented and the mechanisms are beginning to be elucidated. TSG-6 inhibits the inflammatory network of proteases by increasing the inhibitory activity of inter-α-inhibitor and bikunin . TSG-6 also specifically binds and sequesters hyaluronan fragments and has been shown to be a potent inhibitor of neutrophil activation and migration, as well as tissue remodeling , through up-regulation of Cox-2 and prostaglandin D2 expression [28, 29]. The precise mechanism(s) by which TSG-6 protects the lung from acute inflammation remains to be determined.
Knockdown of TSG-6 mRNA expression in hMSCs did not completely abrogate their anti-inflammatory effects (Figure 6) suggesting that additional factors expressed by hMSCs play a role in protecting the lung from LPS-induced inflammation and injury. The hMSCs used in our studies expressed high levels of KGF in the lung (Figure 5b). Lee et al. showed in an isolated, perfused human lung model challenged with endotoxin, that secretion of KGF was essential for the beneficial effect of hMSCs on alveolar epithelial fluid transport . In another study, Aguilar et al. showed that MSCs transfected with a tetracycline-inducible KGF construct partially protected mice from bleomycin-induced pulmonary fibrosis. The mechanisms underlying KGF's protective effect in the lung are not completely understood. KGF has plieotropic effects on alveolar epithelial cells including; mitogenic acitivity, increased surfactant production , inhibition of apoptosis , and increased transcription and/or translation of the major sodium and chloride transport proteins . However, KGF is not known to have direct anti-inflammatory activity. We postulate that TSG-6 and KGF secreted by hMSCs cooperate to attenuate lung injury by suppressing inflammation and promoting lung repair respectively. MSCs secrete other bioactive molecules such as IL1RN (Figure 5c), HGF, EGF, TGF-β1, sTNFR1, Ang1 and STC-1 that may also contribute to their immunomodulatory functions and repair of injured lung [19, 26, 34–38]. Additional experiments are required to determine the relative contribution of each of these factors to the beneficial effects of hMSCs in the lung.
It is tempting to speculate that strategies to enhance hMSC expression of TSG-6 and other beneficial factors such as KGF may improve their lung protective properties. One such strategy is to culture hMSCs as 3D aggregates or as spheroids [39–41]. Recently it was shown that hMSCs cultured in hanging drops formed spheroids and expressed significantly higher levels of TSG-6 and STC-1 compared to hMSCs cultured as monolayers. Moreover, hMSCs from dissociated spheroids were more effective at suppressing inflammation in a mouse model of peritonitis than were hMSCs cultured as adherent monolayers . Using a similar strategy we showed that culture of hMSCs in spheroids increased their expression of TSG-6, STC-1 and KGF (unpublished data). Our finding that hMSCs delivered IP have beneficial effects in the lung suggests the possibility of transplantation of the intact spheroids. We are currently comparing the therapeutic potential of traditionally cultured hMSCs to that of spheroid hMSCs in our animal model of ALI.
This study demonstrates that xenographic transplantation of human MSCs, by multiple routes, attenuates inflammation and lung injury in an in vivo model of acute lung injury. Identification of TSG-6 as a major mediator of the beneficial effects of hMSCs in the lung furthers our understanding of how hMSCs work and supports the concept that the protective effects of MSCs are largely mediated through production of paracrine factors. It is unlikely that a single factor mediates the beneficial effects of hMSCs. MSCs respond to their environment by secreting a variety of factors referred to as the secretome. Elucidation of the hMSC secretome will likely provide strategies to improve the therapeutic potential of hMSCs for treatment of lung injury.
In conclusion, our study provides important preclinical data for potential clinical trials using allogeneic hMSCs for treatment of ALI, a significant source of morbidity and mortality in critically ill patients.
acute lung injury
acute respiratory distress syndrome
human lung fibroblasts
interleukin 1 receptor antagonist
keratinocyte growth factor
multipotent stromal cells
TNF-α-induced protein 6
tumor necrosis factor-α.
The authors are grateful to Ryang Hwa Lee and Hosoon Choi (Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White) and Roxanne Reger (Tulane School of Medicine) for helpful discussions and technical assistance in assaying human MSCs by qRT-PCR and to Krysztof Moroz and Justin Choi (Tulane School of Medicine) for assistance in histological examinations. We are also grateful to Dr. William Wimley for advice on statistical analyses.
This work was supported by the National Institutes of Health Grants P01HL075161 (to BB, DJP, DAW, JAL and DES) and P40RR17447 (DJP). RWB is supported by T32HL007973.
- Erickson SE, Martin GS, Davis JL, Matthay MA, Eisner MD: Recent trends in acute lung injury mortality: 1996-2005. Crit Care Med. 2009, 37: 1574-1579. 10.1097/CCM.0b013e31819fefdf.PubMed CentralView ArticlePubMed
- Rubenfeld GD, Herridge MS: Epidemiology and outcomes of acute lung injury. Chest. 2007, 131: 554-562. 10.1378/chest.06-1976.View ArticlePubMed
- Zambon M, Vincent JL: Mortality rates for patients with acute lung injury/ARDS have decreased over time. Chest. 2008, 133: 1120-1127. 10.1378/chest.07-2134.View ArticlePubMed
- Prockop DJ: Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997, 276: 71-74. 10.1126/science.276.5309.71.View ArticlePubMed
- Bianco P, Robey PG, Simmons PJ: Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell. 2008, 2: 313-319. 10.1016/j.stem.2008.03.002.PubMed CentralView ArticlePubMed
- Uccelli A, Moretta L, Pistoia V: Mesenchymal stem cells in health and disease. Nat Rev Immunol. 2008, 8: 726-736. 10.1038/nri2395.View ArticlePubMed
- Prockop DJ, Kota DJ, Bazhanov N, Reger RL: Evolving paradigms for repair of tissues by adult stem/progenitor cells (MSCs). J Cell Mol Med. 2010, 14: 2190-2199. 10.1111/j.1582-4934.2010.01151.x.PubMed CentralView ArticlePubMed
- ClinicalTrials.gov. [http://clinicaltrials.gov]
- Sueblinvong V, Weiss DJ: Stem cells and cell therapy approaches in lung biology and diseases. Transl Res. 2010, 156: 188-205. 10.1016/j.trsl.2010.06.007.PubMed CentralView ArticlePubMed
- Caplan AI, Dennis JE: Mesenchymal stem cells as trophic mediators. J Cell Biochem. 2006, 98: 1076-1084. 10.1002/jcb.20886.View ArticlePubMed
- Ren G, Su J, Zhang L, Zhao X, Ling W, L'Huillie A, Zhang J, Lu Y, Roberts AI, Ji W, Zhang H, Rabson AB, Shi Y: Species variation in the mechanisms of mesenchymal stem cell-mediated immunosuppression. Stem Cells. 2009, 27: 1954-1962. 10.1002/stem.118.View ArticlePubMed
- Bai L, Lennon DP, Eaton V, Maier K, Caplan AI, Miller SD, Miller RH: Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune response and promote endogenous repair in animal models of multiple sclerosis. Glia. 2009, 57: 1192-1203. 10.1002/glia.20841.PubMed CentralView ArticlePubMed
- Gordon D, Pavlovska G, Glover CP, Uney JB, Wraith D, Scolding NJ: Human mesenchymal stem cells abrogate experimental allergic encephalomyelitis after intraperitoneal injection, and with sparse CNS infiltration. Neurosci Lett. 2008, 448: 71-73. 10.1016/j.neulet.2008.10.040.PubMed CentralView ArticlePubMed
- Institute for Regenerative Medicine, Texas A&M Health Sciences Center: [http://medicine.tamhsc.edu/irm/msc-distribution.html]
- Lakatos HF, Burgess HA, Thatcher TH, Redonnet MR, Hernady E, Williams JP, Sime PJ: Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation. Exp Lung Res. 2006, 32: 181-199. 10.1080/01902140600817465.View ArticlePubMed
- Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, Semprun-Prieto L, Delafontaine P, Prockop DJ: Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell. 2009, 5: 54-63. 10.1016/j.stem.2009.05.003.PubMed CentralView ArticlePubMed
- Clopper C, Pearson E: The use of confidence or fiducial limits illustrated in the case of the binomial. Biometrika. 1934, 26: 404-413. 10.1093/biomet/26.4.404.View Article
- JavaStat: Exact Binomial and Poisson Confidence Intervals. [http://statpages.org/confint.html]
- Gupta N, Su X, Popov B, Lee JW, Serikov V, Matthay MA: Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice. J Immunol. 2007, 179: 1855-1863.View ArticlePubMed
- Milner CM, Higman VA, Day AJ: TSG-6: a pluripotent inflammatory mediator?. Biochem Soc Trans. 2006, 34: 446-450. 10.1042/BST0340446.View ArticlePubMed
- Wisniewski HG, Vilcek J: Cytokine-induced gene expression at the crossroads of innate immunity, inflammation and fertility: TSG-6 and PTX3/TSG-14. Cytokine Growth Factor Rev. 2004, 15: 129-146. 10.1016/j.cytogfr.2004.01.005.View ArticlePubMed
- Lee JW, Fang X, Gupta N, Serikov V, Matthay MA: Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung. Proc Natl Acad Sci USA. 2009, 106: 16357-16362. 10.1073/pnas.0907996106.PubMed CentralView ArticlePubMed
- Ortiz LA, Dutreil M, Fattman C, Pandey AC, Torres G, Go K, Phinney DG: Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci USA. 2007, 104: 11002-11007. 10.1073/pnas.0704421104.PubMed CentralView ArticlePubMed
- Mei SH, McCarter SD, Deng Y, Parker CH, Liles WC, Stewart DJ: Prevention of LPS-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1. PLoS Med. 2007, 4: e269-10.1371/journal.pmed.0040269.PubMed CentralView ArticlePubMed
- Xu J, Woods CR, Mora AL, Joodi R, Brigham KL, Iyer S, Rojas M: Prevention of endotoxin-induced systemic response by bone marrow-derived mesenchymal stem cells in mice. Am J Physiol Lung Cell Mol Physiol. 2007, 293: L131-141. 10.1152/ajplung.00431.2006.View ArticlePubMed
- Nemeth K, Keane-Myers A, Brown JM, Metcalfe DD, Gorham JD, Bundoc VG, Hodges MG, Jelinek I, Madala S, Karpati S, Mezey E: Bone marrow stromal cells use TGF-beta to suppress allergic responses in a mouse model of ragweed-induced asthma. Proc Natl Acad Sci USA. 2010, 107: 5652-5657. 10.1073/pnas.0910720107.PubMed CentralView ArticlePubMed
- Forteza R, Casalino-Matsuda SM, Monzon ME, Fries E, Rugg MS, Milner CM, Day AJ: TSG-6 potentiates the antitissue kallikrein activity of inter-alpha-inhibitor through bikunin release. Am J Respir Cell Mol Biol. 2007, 36: 20-31.PubMed CentralView ArticlePubMed
- Milner CM, Day AJ: TSG-6: a multifunctional protein associated with inflammation. J Cell Sci. 2003, 116: 1863-1873. 10.1242/jcs.00407.View ArticlePubMed
- Mindrescu C, Le J, Wisniewski HG, Vilcek J: Up-regulation of cyclooxygenase-2 expression by TSG-6 protein in macrophage cell line. Biochem Biophys Res Commun. 2005, 330: 737-745. 10.1016/j.bbrc.2005.03.040.View ArticlePubMed
- Aguilar S, Scotton CJ, McNulty K, Nye E, Stamp G, Laurent G, Bonnet D, Janes SM: Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis. PLoS One. 2009, 4: e8013-10.1371/journal.pone.0008013.PubMed CentralView ArticlePubMed
- Yano T, Mason RJ, Pan T, Deterding RR, Nielsen LD, Shannon JM: KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in adult rat lung. Am J Physiol Lung Cell Mol Physiol. 2000, 279: L1146-1158.PubMed
- Ware LB, Matthay MA: Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair. Am J Physiol Lung Cell Mol Physiol. 2002, 282: L924-940.View ArticlePubMed
- Wang Y, Folkesson HG, Jayr C, Ware LB, Matthay MA: Alveolar epithelial fluid transport can be simultaneously upregulated by both KGF and beta-agonist therapy. J Appl Physiol. 1999, 87: 1852-1860.PubMed
- Block GJ, Ohkouchi S, Fung F, Frenkel J, Gregory C, Pochampally R, DiMattia G, Sullivan DE, Prockop DJ: Multipotent stromal cells are activated to reduce apoptosis in part by upregulation and secretion of stanniocalcin-1. Stem Cells. 2009, 27: 670-681.View ArticlePubMed
- Fang X, Neyrinck AP, Matthay MA, Lee JW: Allogeneic human mesenchymal stem cells restore epithelial protein permeability in cultured human alveolar type II cells by secretion of angiopoietin-1. J Biol Chem. 2010, 285: 26211-26222. 10.1074/jbc.M110.119917.PubMed CentralView ArticlePubMed
- Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, Mu H, Melo LG, Pratt RE, Ingwall JS, Dzau VJ: Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J. 2006, 20: 661-669. 10.1096/fj.05-5211com.View ArticlePubMed
- Katsha AM, Ohkouchi S, Xin H, Kanehira M, Sun R, Nukiwa T, Saijo Y: Paracrine factors of multipotent stromal cells ameliorate lung injury in an elastase-induced emphysema model. Mol Ther. 2010, 19: 196-203.PubMed CentralView ArticlePubMed
- Yagi H, Soto-Gutierrez A, Kitagawa Y, Tilles AW, Tompkins RG, Yarmush ML: Bone marrow mesenchymal stromal cells attenuate organ injury induced by LPS and burn. Cell Transplant. 2010, 19: 823-830. 10.3727/096368910X508942.PubMed CentralView ArticlePubMed
- Frith JE, Thomson B, Genever PG: Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng Part C Methods. 2009, 16: 735-749.View Article
- Potapova IA, Brink PR, Cohen IS, Doronin SV: Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells. J Biol Chem. 2008, 283: 13100-13107. 10.1074/jbc.M800184200.PubMed CentralView ArticlePubMed
- Qihao Z, Xigu C, Guanghui C, Weiwei Z: Spheroid formation and differentiation into hepatocyte-like cells of rat mesenchymal stem cell induced by co-culture with liver cells. DNA Cell Biol. 2007, 26: 497-503. 10.1089/dna.2006.0562.View ArticlePubMed
- Bartosh TJ, Ylostalo JH, Mohammadipoor A, Bazhanov N, Coble K, Claypool K, Lee RH, Choi H, Prockop DJ: Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci USA. 2010, 107: 13724-13729. 10.1073/pnas.1008117107.PubMed CentralView ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.